Detection and analysis of poly(amino acids) is of great importance in a multitude of diverse activities, ranging from commercial enzyme production, forensics analysis and diagnostics to basic research in biochemistry, molecular biology, neuroscience, developmental biology or physiology. As used herein, a poly(amino acid) is any homopolymer or heteropolymer of amino acids, including peptides and proteins. Primarily, poly(amino acids) are detected and characterized using gel electrophoresis, by solution quantitation assays or by detection on solid supports, such as filter membranes.
Electrophoresis of poly(amino acids) is most commonly carried out using polyacrylamide gels. Unmodified protein or other poly(amino acid) bands in gels are generally not visible to the naked eye. Thus, for electrophoretic gels to be useful, the bands or spots must be stained, so that they can be localized and identified. Two of the most common methods of staining poly(amino acids) on gels are COOMASSIE Brilliant Blue staining (hereafter referred to as CBB staining) and silver staining.
For CBB staining, the electrophoresis gel is first fixed, stained for several hours with a triphenylmethane-based dye, then destained for several more hours. The resulting stained gel is pale blue with dark blue bands containing the poly(amino acids). The sensitivity of CBB staining is strongly dependent on how thoroughly the gel is destained. A destaining period of 24 hours typically allows as little as 0.03-0.1 .mu.g of poly(amino acid) to be detected in a single band. However, excessive destaining also results in signal loss from the bands. Although CBB staining is inexpensive, easy to use, and the resulting gels are easily preserved, CBB staining gives linear responses within only a narrow dynamic range. Furthermore, once stained using CBB, the poly(amino acids) in the gel cannot be blotted for immunoanalysis, CBB staining is somewhat selective for poly(amino acid) composition and tends to bind small peptides poorly.
Silver staining utilizes the differential reduction of silver ions bound to the side chains of amino acids in poly(amino acids). For particular poly(amino acids), silver staining is approximately 100- to 1000-fold more sensitive than CBB staining and is capable of detecting 0.1-1 ng of poly(amino acid) in a single band. A gel that has been silver stained is clear to yellow-tan, with gray, dark-brown or black poly(amino acid) bands. Silver stained gels can readily preserved, as for CBB stained gels. Like CBB staining, silver staining is time-consuming and yields a narrow linear response for densitometric quantitation. Also, the stained gels cannot be blotted for further analysis. In addition, silver staining requires the handling of several very toxic, unstable and expensive solutions, and the resulting staining is extremely selective for poly(amino acid) composition, both in band color and band intensity. Finally, silver staining requires an exacting methodology that is often difficult to perform reproducibly.
A relatively recent method for staining protein gels uses the dye Nile red (9-diethylamino-5H-benzo(.alpha.)phenoxazine-5-one) as a fluorescent stain for the poly(amino acid) bands in SDS-polyacrylamide gels (Daban et al., ANAL. BIOCHEM. 199, 169 (1991)). The use of Nile red as a stain for poly(amino acids) on gels is rapid, does not require that the gel be fixed prior to staining, and the resulting stained gels can be blotted for further analysis. However, Nile red-stained gels photobleach rapidly, requiring gels to be documented immediately with photography. The use of Nile red in combination with black and white POLAROID photography is capable of detecting 30 ng of poly(amino acid) in a single band, putting its sensitivity between that of CBB staining and silver staining. Finally, Nile red itself is very insoluble, resulting in poor penetration of gel bands and making staining solutions difficult to handle,
Use of the present invention possesses many advantages over known methods for staining poly(amino acids) on gels: Staining is very rapid, and is relatively insensitive to poly(amino acid) composition. Visualization of stained gels is possible without destaining, and the stained bands remain readily detectable for several days. The dyes used in the current method are readily soluble and stable in aqueous staining solutions. In addition, the dyes exhibit a large Stokes shift between the absorbance and emission maxima. Finally, the staining procedure of the present invention is rapid and simple, requires minimal labor, and allows the detection of as little as 1 ng of poly(amino acid) per band; this sensitivity is in many cases equal to or better than that of silver staining, with far less hazard and expense, and is more than an order of magnitude better than CBB or Nile red staining.
The dyes of the present invention can also be used to detect poly(amino acids) on filter membranes or other solid supports, or in solution. The use of the dyes of the current invention for staining poly(amino acids) in solution can be used to quantitate poly(amino acids) with greater sensitivity than other known methods (Table 3), including absorbance-based methods, such as the Lowry method (J. BIOL. CHEM., 193, 265 (1951)), the bicinchoninic acid (BCA) method (ANAL, BIOCHEM., 150, 76 (1985)), and the Bradford method (ANAL. BIOCHEM., 72, 248 (1976)); and fluorescence-based methods, such as those employing Nile red (ANAL. BIOCHEM, 199, 162 (1991)). Using preferred embodiments of this new solution assay, the linear dynamic range for quantitation extends over almost three orders of magnitude in poly(amino acid) concentration (from about 30 ng/mL to about 10 .mu.g/mL, see Table 2), in contrast to the more limited range of the well-known Lowry (0.1-2 mg/mL), Bradford (0.2-1.4 mg/mL), and BCA (10-2000 .mu.g/mL) methods. The solution assay of the present invention is simple and suitable for use with either standard fluorometers or automated microtiter plate readers, or can be modified for use with electrophoretic capillaries or density gradients.
The preparation and characterization of the merocyanine dyes of the present invention has been well documented. A large number of useful styryl merocyanine dyes (commonly referred to as RH dyes) have been previously prepared by Rina Hildesheim (Grinvald et al., BIOPHYS. J. 39, 301 (1982), incorporated by reference), Leslie Loew (Loew et al., J. ORG. CHEM. 49, 2546 (1984), incorporated by reference) and others, as useful probes for measuring electric potentials in cell membranes. Useful membrane potential measurements only occur in live cells and artificial liposomes, where the fluorescence intensity of a suitable dye as it is associated with the membrane changes as the membrane is subjected to an electrical gradient. In addition to the above membrane potential probes, an extensive variety of other merocyanine dyes have been described by Brooker et al. (J. AM. CHEM. SOC. 73, 5326 (1951), incorporated by reference), primarily for use in the photographic industry, although Brooker et al. do not describe the fluorescence properties of the merocyanines. The present invention describes the use of these and other reagents to label and detect poly(amino acids), including cellular components, outside of the cellular milieu. This novel use of merocyanine dyes, which is faster, easier, less expensive, and less hazardous than other known methods of staining poly(amino acids), is neither anticipated nor obvious from previously described uses.